Throughout this application, various publications are referenced in parentheses by arabic numbers. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Cancer is the second leading cause of death in the United States. When cancer has metastasized, it can only be cured by systemic therapy, usually cytotoxic chemotherapy. However, it is often true that in this case chemotherapy is ineffective. There are currently attempts to develop new anti-cancer agents, including antisense oligonucleotides. Antisense oligonucleotides can specifically inhibit the translation of mRNA into protein. However, their use as therapeutic agents is limited because of intracellular transport and compartmentalization problems.
Thus, there are difficulties associated with delivery of antisense oligonucleotides to their targets. At least in tissue culture, antisense oligonucleotides almost invariably must be condensed with a delivery reagent to ensure adequate cellular uptake and release from sequestered sites in the endosomes/lysosomes. The most commonly employed delivery reagents are cationic lipids (e.g., Lipofectin), but these reagents may contribute their own cytotoxic effects, which affects the phenotype produced after treatment of the cells with the antisense effector molecule.
Accordingly, a large number of peptide delivery vehicles for antisense oligonucleotides have been devised. There are at least 20 peptides that increase the delivery of oligonucleotides to cells. These include pH sensitive fusogenic peptides, Antennapedia-type peptides, and the HIV tat C-terminus peptide. Other peptides that have been covalently conjugated to oligonucleotides include the ER-retaining peptide YKDEL (1) (SEQ ID NO:8), and various nuclear localization signal peptides (including the PKKKRKV sequence, SEQ ID NO:9, derived from the SV40 large-T antigen (2,3,4,5).
Non-covalent peptide oligonucleotide complexes have also been employed to increase cellular delivery (reviewed in 6). Morris, et al. (7) used a 27-mer peptide, called MPG, which was composed of the N-terminal domain of the HIV gp41 fusion sequence fused to the C-termain domain derived from the nuclear localization signal derived from the SV40 large-T antigen has. Nuclear localization of oligonucleotide in fibroblasts was observed. Pichon, et al. (11) employed a permeabilizing peptide derived ultimately from an analog of the N-terminal sequence of the HA2 subunit of the influenza virus hemeaglutinin. Permeabilization was successful as judged by the nuclear localization of a fluoresceinated oligonucleotide. Pichon et al. (12) subsequently employed histidylated oligolysines to deliver antisense oligonucleotides targeted to ICAM-1, and demonstrated excellent antisense activity. However, in almost all the examples of peptide-mediated delivery given above, it is unclear if any sequence specific downregulation of the expression of a target mRNA was observed. An effective non-lipidic way of reliably delivering functioning antisense oligonucleotides is still sought.